Microbial transformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus

The biotransformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus MT 5.3 yielded a rare derivative that was elucidated by spectrometric methods. The fungus led to the formation of a different product through an unusual epoxidation reaction between C4 and C5, formation of a C3,C10 ether bridge, and a methoxylation of the C1 of tagitinin C. The chemical structure of the product, namely 1 beta-methoxy-3 alpha-hydroxy-3,10 beta-4,5 alpha-diepoxy-8 beta-isobutyroyloxygermacr-11(13)-en-6 alpha,12-olide, is the same as that of a derivative that was recently isolated from the flowers of a Brazilian population of Mexican sunflower (Tithonia diversifolia), which is the source of the substrate tagitinin C. The in vitro cytotoxic activity of the substrate and the biotransformed product were evaluated in HL-60 cells using an MTT assay, and both compounds were found to be cytotoxic. We show that soil fungi may be useful in the biotransformation of sesquiterpene lactones, thereby leading to unusual changes in their chemical structures that may preserve or alter their biological activities, and may also mimic plant biosynthetic pathways for production of secondary metabolites.

Chemoenzymatic approaches to obtain chiral-centered selenium compounds

The synthesis of chiral-centered selenium compounds is presented. Enantioselective oxidations of these organoselenium compounds were performed using a wide range of biocatalysts, including Baeyer-Villiger monooxygenases, oxidoreductases-containing Aspergillus terreus and lipase (Cal-B) in the presence of oxidants. Finally, efficient synthesis of enantiopure organoselenium compounds using a kinetic resolution approach mediated by Cal-B was achieved. (C) 2012 Elsevier Ltd. All rights reserved.

Exploiting the enzymatic machinery of Arthrobacter atrocyaneus for oxidative kinetic resolution of secondary alcohols

We evaluated Arthrobacter atrocyaneus (R1AF57) as producer of oxidoreductases for oxidative kinetic resolution of racemic secondary alcohols via oxidation reaction. This bacterium was isolated from Amazon soil samples using medium enriched with (RS)-1-(4-methylphenyl)ethanol as a carbon source. The kinetic resolution of several secondary alcohols through enantioselective oxidation mediated by resting cells and growing cells of A. atrocyaneus was efficiently achieved for the most alcohols. In general, it was possible to obtain only the (S)-enantiomer from (RS)-1-arylethanols.

Production of a xylose-stimulated beta-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of beta-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 +/- A 411.2 U g(-1), while beta-glucosidase production was increased about 2.6-fold, reaching 20.7 +/- A 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis beta-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for beta-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The beta-glucosidase maintained about 95 % of its activity after 26 h in water at 55 A degrees C, with half-lives of 15.7 h at 60 A degrees C and 5.1 h at 65 A degrees C. The presence of xylose during heat treatment at 65 A degrees C protected beta-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 A degrees C. Xylose stimulated beta-glucosidase activity up to 1.7-fold, at 200 mmol L-1. The notable features of both xylanase and beta-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Marine Fungi Aspergillus sydowii and Trichoderma sp Catalyze the Hydrolysis of Benzyl Glycidyl Ether

Whole cells of the marine fungi Aspergillus sydowii Gc12, Penicillium raistrickii Ce16, P. miczynskii Gc5, and Trichoderma sp. Gc1, isolated from marine sponges of the South Atlantic Ocean (Brazil), have been screened for the enzymatic resolution of (+/-)-2-(benzyloxymethyl)oxirane (benzyl glycidyl ether; 1). Whole cells of A. sydowii Gc12 catalyzed the enzymatic hydrolysis of (R,S)-1 to yield (R)-1 with an enantiomeric excess (ee) of 24-46% and 3-(benzyloxy)propane-1,2-diol (2) with ee values < 10%. In contrast, whole cells of Trichoderma sp. Gc1 afforded (S)-1 with ee values up to 60% and yields up to 39%, together with (R)-2 in 25% yield and an ee of 32%. This is the first published example of the hydrolysis of 1 by whole cells of marine fungi isolated from the South Atlantic Ocean. The hydrolases from the two studied fungi exhibited complementary regioselectivity in opening the epoxide ring of racemic 1, with those of A. sydowii Gc12 showing an (S) preference and those of Trichoderma sp. Gc1 presenting an (R) preference for the substrate.

Aspergillus fumigatus calcineurin interacts with a nucleoside diphosphate kinase

The Ca2+-calcineurin pathway affects virulence and morphogenesis in filamentous fungi. Here, we identified 37 CalA-interacting proteins that interact with the catalytic subunit of calcineurin (CalA) in Aspergillus fumigatus, including the nucleoside diphosphate kinase (SwoH). The in vivo interaction between CalA and SwoH was validated by bimolecular fluorescence complementation. A. fumigatus swoH is an essential gene. Therefore, a temperature-sensitive conditional mutant strain with a point mutation in the active site, SwoH(V83F), was constructed, which demonstrated reduced growth and increased sensitivity to elevated temperatures. The SwoH(V83F) mutation did not cause a loss in virulence in the Galleria mellonella infection model. Taken together these results imply that CalA interacts with SwoH. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Molecular characterization by amplified fragment length polymorphism and aflatoxin production of Aspergillus flavus isolated from freshly harvested peanut in Brazil

Brazil contributes substantially to the global peanut production, and the state of Sao Paulo is the largest producer in the country. Peanut crops can be contaminated by Aspergillus flavus strains producing aflatoxins, which are highly toxic and carcinogenic. Thus, the production of high-quality peanuts is crucial both for the commercial peanut industry and as a matter of public health. In this study, we used amplified fragment length polymorphism analysis (AFLP) to investigate the genetic variability among A. flavus strains isolated from fresh peanuts harvested in four different regions in the state of Sao Paulo, and to determine whether the molecular genetic profiles correlated with aflatoxin production or sclerotia formation. AFLP analysis generated 78 fragments ranging from 27 to 365 base pairs in length. Thirteen percent were not polymorphic. Genotyping identified twelve groups of A. flavus. On the basis of the polymorphisms identified, similarity between the isolates ranged from 37% to 100%. Of all isolates collected, 91.7% produced aflatoxins and 83.9% produced small sclerotia. Statistical analysis failed to suggest any relationship between the presence of sclerotia and mean levels of aflatoxins B-1 and B-2. Furthermore, a dendrogram based on AFLP data revealed substantial genetic variability among the A. flavus strains, but showed no correlation between dendrogram groups separated by molecular genetic features and production of aflatoxins B-1 or B-2 or the formation of sclerotia.

Production of Fructooligosaccharides by Aspergillus phoenicis Biofilm on Polyethylene as Inert Support

Aspergillus phoenicis biofilms on polyethylene as inert support were used to produce fructooligosaccharides (FOS) in media containing 25% (m/V) of sucrose as a carbon source. The maximum production of total FOS (122 mg/mL), with 68% of 1-kestose and 32% of nystose, was obtained in Khanna medium maintained at 30 degrees C for 48 h under orbital agitation (100 rpm). At high concentrations of sucrose (30%, m/V), the recovery of FOS was higher than that observed at a low concentration (5%, m/V). High levels of FOS (242 mg/mL) were also recovered when using the biofilm in sodium acetate buffer with high sucrose concentration (50%, m/V) for 10 h. When the dried biofilm was reused in a fresh culture medium, there was a recovery of approx. 13.7% of total FOS after 72 h of cultivation at 30 C, and 10% corresponded to 1-kestose. The biofilm morphology, analyzed by scanning electron microscope, revealed a noncompact mycelium structure, with unfilled spaces and channels present among the hyphae. The results obtained in this study show that A. phoenicis biofilms may find application for FOS production in a single-step fermentation process, which is cost-effective in terms of reusability, downstream processing and efficiency.

Strain-specific polyketide synthase genes of Aspergillus niger

In silico comparison of 34 putative pks genes in Aspergillus niger strain CBS 513.88 versus A. niger strain ATCC 1015 genome revealed significant nucleotide identity (>95% covering a minimum of 99% of the gene sequence) for 31 of these genes (approximately 91%). A. niger CBS 513.88 harbors three putative pks genes (An01g01130, An11g05940, and An15g07920), for which nucleotide identity was not found in A. niger ATCC 1015. To compare the results of the in silico analysis with the in vivo situation, experimental data were obtained for a large number of A. niger strains obtained from different substrates and geographical regions. Three putative Os genes that were found to be variable between the two A. niger strains using bioinformatics tools were in fact strain-specific genes based on experimental data. The PCR amplification signals for the An01g01130, An11g05940, and An15g07920 pks genes were detected in only 97%, 71%, and 26% of the strains, respectively. Southern blot analyses confirmed the PCR data. Because one of the strain-specific pits genes (An15g07920) is located in a putative ochratoxin cluster, we focused our investigation on that region. We assessed the ochratoxin production capability of the 119 A. niger strains and found a positive association between the presence of this pia gene and the capability of the respective strain to produce ochratoxin. (C) 2012 Elsevier B.V. All rights reserved.

Characterization of aflatoxigenic and non-aflatoxigenic strains of Aspergillus section Flavi isolated from corn grains of different geographic origins in Brazil

Aflatoxins can cause great economic losses and serious risks to humans and animals health. The largest aflatoxin producers belong to Aspergillus section Flavi and can occur naturally in food commodities. Studies showed that molecular tools as well as the type of sclerotia produced by the strains could be helpful for identification of Aspergillus species and could be correlated with levels of toxin production. The purpose of this work was to characterize the genetic diversity using AFLP technique, the type of sclerotia and the ability of aflatoxin production by isolated strains from corn of different origins in Brazil, and to verify whether qPCR based on aflR and aflP genes is appropriate for estimating the level of aflatoxin production. All the 75 strains were classified as A. flavus and the AFLP technique showed a wide intraspecific variability within them. Regarding sclerotia production, 34% were classified as S and 66% as L type. Among the aflatoxin-producers, 52.8% produced aflatoxin B-1, while 47.2% aflatoxins B-1 and B-2. Statistical analysis showed no correlation between sclerotia production and aflatoxigenicty, and no correlation between the phylogenetic clusters and aflatoxin production. Concerning the relative expression of aflR and aflP, Pearson's correlation test demonstrated low positive correlation between the expression of the aflR and aflP genes and the production of AFB(1) and AFB(2), but showed high positive correlation between aflR and aflP expression. In contrast to the other reference strains, A. oryzae ATCC 7282 showed no amplification of aflR and aflP. The results highlight the need for detection of reliable and reproducible markers with a high positive correlation with aflatoxin production.

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen micro-organisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS: In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION: Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages. Copyright (c) 2012 Society of Chemical Industry

Immobilization of a recombinant endo-1,5-arabinanase secreted by Aspergillus nidulans strain A773

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 degrees C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni2+, glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 degrees C. The half-life of glyoxyl 4% derivative at 60 degrees C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose. (C) 2012 Elsevier B.V. All rights reserved.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

Xylanase and beta-Xylosidase Production by Aspergillus ochraceus: New Perspectives for the Application of Wheat Straw Autohydrolysis Liquor

The xylanase biosynthesis is induced by its substrate-xylan. The high xylan content in some wastes such as wheat residues (wheat bran and wheat straw) makes them accessible and cheap sources of inducers to be mainly applied in great volumes of fermentation, such as those of industrial bioreactors. Thus, in this work, the main proposal was incorporated in the nutrient medium wheat straw particles decomposed to soluble compounds (liquor) through treatment of lignocellulosic materials in autohydrolysis process, as a strategy to increase and undervalue xylanase production by Aspergillus ochraceus. The wheat straw autohydrolysis liquor produced in several conditions was used as a sole carbon source or with wheat bran. The best conditions for xylanase and beta-xylosidase production were observed when A. ochraceus was cultivated with 1% wheat bran added of 10% wheat straw liquor (produced after 15 min of hydrothermal treatment) as carbon source. This substrate was more favorable when compared with xylan, wheat bran, and wheat straw autohydrolysis liquor used separately. The application of this substrate mixture in a stirred tank bioreactor indicated the possibility of scaling up the process to commercial production.

The fungal metabolite eugenitin as additive for Aspergillus niveus glucoamylase activation

Endophytic microorganisms live inside tissues of host plants apparently do not causing warning to them, and area promising source of bioactive molecules as antimicrobial and antitumoral drugs. In this work, we report the isolation of eugenitin from cultures of the endophyte Mycoleptodiscus indicus and its potential as additive for Aspergillus niveus glucoamylase activation. The glucoamylase hydrolytic activity increased twofold using 5 mM of eugenitin and this activation could be explained by the binding mode of eugenitin with the three-dimensional structure of glucoamylase. The in silica prediction of ligand binding sites revealed at least 9 possible interaction sites able to accommodate eugenitin on glucoamylase from Hypocrea jecorina. Besides, we evaluated the effect of pH and temperature on activity and stability, as well as in the hydrolysis of different substrates and kinetic parameters either in presence or absence of eugenitin. The results displayed by eugenitin as additive to glucoamylase activation are promising and provide novel perspectives for applications of fungal metabolites. (C) 2011 Elsevier B.V. All rights reserved.